Improving the growth of mushroom mycelium



Patented Feb. 6, 1940 mirnovrNq- "the GR I MYCELIUM ow'r'n Ioinusnnoorrf Benjamin Stoller, Coatesvilla'lia ascignor to Louis F.Lambert .Qoatesvllle, Pa-.

No Drawing. iam'ni SerialNo. 138,368 l 4 Claims,

This invention relatesto a method of treating k substrate for improvingthe growth. of the mushroom mycelium both in the 'rnanuia'ctur oi;spawnand in the production 7 of mushroomi-spo'rophorejs,

By the inethodiri accordance-with thisinven; tion the, mycelium growsmore abundantly," is" denser and l more vigorous-because the substrateis made moresuitablefor growth;

As a result of this invention it is now possible for the mycelium togrow completely through the sterilized substrate in the production ofspawn! only the white mycelial growth is evident,'so that the spawnbottle-,has awhitefmilky appearance. whereas in the ordinary spawnbottl'ethe spawn appears reddish, speckled with white, on account oi"only apa'ttial growth of the mycelium through the substrate.Furthermore, strand growth of spawn is entirelyeliminated'.

7 By the method in--accordancewxth this invents, Q

tion, the mycelium grows morequickly and more vigorously Jinthe'mushroom bed, as, observedbyi the larger concen'trationgof carbondioxide 7 evolved. produces mushrooms earlienfa larger yield ofmushrooms.

,It is known-that the mushroom mycelium, prond gives a duces organicacids, such as oxalic and carbonic aci'ds,-whenit grows through-thesubstrate. If

these acids are not removed, 'as'by" precipitation,

oxidation or gaseb'us evolution, they "will soon I make the-mediumaorsubstrate unsuitable for a further mycelial growth. If the mycelialgrowth on ordinary washed and sterilized .cornposted manure is observedcarefully, it maybe seen that the mushroom hyphae have only part iallypenetrated this medium and-thatthe manure has assumed a reddish hue,,This red color has been definitely correlated ,with' a decrease in thepH i value of the substrate; that is, the H ion concentration hasbe'enincreased by some acids formed in the coursecf the; growth of the"mushroom mycelium, and the pH value iscorrespondingly decreased. Theseacids-which are either formed.

favorable for the complete penetration'by the mushroom hyphae. In suchcase,' the substrate appears reddish and speckled withpthe whitemycelium instead of being completely white and, dense, as would havebeen 'the case if the cation April 22, 193i,

myceiiumhad completelyor' more completely "penetrated" the substrate andhofree acids pre- 1 ,v'ailed. a Y The pres'entinventiontherefore'comprises the method and means whereby this acid; normallyarising; from the mycelial growth," may be "new" fecting thesubstrategor making it 'unsuitable for." the initial growthof the"spawn.

It was found that by addition to the substrate] 10 of an alkaline' earthsubstancasuch asca hyv droxideonto a less extent, Mg hydroxide inqua'ntity sufflcientto neutralize the said acid condition of the substrate,would make the substrate 'injuri'ous'to the initial growth ofthe'spawn.For

example, if 4% to 6%lime is addedtowash'ed jhorse manure, with a. pH of7.6, the pH of the mixture after sterilization becomes 8.8 to 9.4 asshown in Table 1. ;At this ,high pH valuethep myc'eliinn usually failsto grow. I l

the lime with or without the' addition "or a butiering salt on the pH oiwashed horse manure. The manure use'd'in this instance was'ionlysiizhtlycomposted and so :had'an initial pH'ot 71am Y ."stead of theusualpH'8L0of manure better com postedeglBut even at this low initialpH, the pH of the manurerose to 8.3 when, only 11% lime was Y added.When. 4% ,to,,6% ,limeis added, that is} thefquantity of lime necessaryforthe most 30 abundant growthof the myceliunijthe pH of the substratebecomes entirely unsatisfactory for the growth of the'mycelium.However,when such chemicals as .-Al, Fe, or Mn are added, the pH may beadjustedas desired, irrespective of how much lime is added: \ILinsteadof adding equal amounts of the lime and theAl- Fe or Mn salts, more ofthe latterare added; the pH may be held the same as that for the, check.

Al, Fe, Mn, Cr, and Zn are not the only elemerits of salts whichwillantagonize or butler the eiiect of alkali or the alkaline earthelements; Any salt or substance which will increase the H ionconcentration of the substrate and not in itself be poisonous tothemushroom mycelium tralized,without,atthesame timeginjuriouslyaf- InTable i'is shownisoinedataon the eflect of r w alkaline earth elements.The benefits derived from the common practice of adding soil .to manure,when composting, may be to supply such elements as ALFe, Mn, Ca, Mg,etc. present in density or the mycelial growth, or the complete- .nessofthe penetration of the substrate seems to depend rather on the presenceof certain substances, as shown by my method, which will pre- 6 the5011. According to my method these elements cipitate, oxidize, orprevent metabolic end-prodare deliberately added in a highly availabletorm'. ucts or deleterious transformations in the sub- TAnnn 1.Theeffect of hydratcdlime with aaaiwithoat the addmon of aluminum, tron,

and manganese on the pH ofaz rsh'ed composted ho'rse'manure, pH 7.6,after the micfare of c emicale and manure hadteen sterilized at 15 lbs.pressure for 4 hours. The pH was determined with the glass electrode. l0

Hydrated lime Lime plus Al andFe salts Lime plus MnBO.

Lime Lime Percent FeSOi Al (S04)u MnSO; arm on dry pH grams gramsPercent DH grams Percent pH basis Grams on dry Grams on dry basis basisI All chemicals except Mn (MnSO4, 2H2O, C. P.) strate during the courseof growth. There may, were of the technical grade as used in actualplant however. exist certain optimum pH values for 8. operations.specific medium, especially in the absence of 9. TABLE 2."l'he efl'ectof chemicals on the H, rlensit and extent of mycelial growth.

The pH was'meuxured with the glans e ee rode. he extent of'growth wasestimated by measuring with a flexible ruler the surface of the spawnbottle in sq. cm. where the myceli'um had not grown: Zero would indicatea full grown bottle, and Fifty a half rown bottle. Growth was measuredafter 27 days. 30

Salts added to washed manure, percent dry basis :0. cf lpHlicieitore pHof 2223: ga 'ifgg ott e noca Ion spawn a5 Sans Percent basis of 10 basisof 100 Check 8.0 6.4 3 8 Hydrate 2 8. 6 6. 8 4 50 ..d0.. 4 8.8 7.2 6 60l} 9.1 Nogrowth Calcium carbonate. U. 6 8. 3. 7. 2' 5 B 40 do s 8.3 1.2s s 40 Calcium sulphate, C. P. 4 7.0 5.7 3 0 do (l 6.9 5.7 3 0 8,Calcium nitrate, tech. 2 7.1 5. 9 2 5 do 3 6.9 5.9 2 -5 10 Magnesiumsulphate, U. S. P 4 7.2 6.2 3 0 l1. Ferric sulphate, C. P 2 7. 1 6. l 20 ...d0 2

l. 6 15.. Aluminum sulphate. tecl1. l E "2""'C ;m; lme percen a ec 16.Aluminum sulphate, tech 3 Ferrous sulphate. tech 3 me 7 l7 Aluminumsulphate. 3

Ferrous sulphate... 3 Li R 1 t me I 21 Zinc sulphate, C. IR. 4 50 ..do.6 :Lime. 6 23 Aluminum sulphate, tech 2 Potassium carbonate. l 24.Aluminum sulphate 2 Potassium carbonate... 2 r v 25 Aluminum sulphate...2 g5 Potas ium carbonate. 3 Blight growth do 0. 22 i. No growth It hasnot been possible to set any pH or any sufliciency of v a. calcium saltproperly bufiered.

' narrow range as the optimum value for, mycelial It has been reportedin the literatureiFreargjc growth in the presenceiof a sufllciency 01such Styer, Haley-PLphysioL 3: 91-4, 1928) that the substancesas acalcium salt properly buffered. best growth (1. e. most rapid) obtainedin cultures- 'Ifhe mushroom .mycelium has been round to grow. rapidlyanywhere on the pH range from 6.9

was nearest to a pH at 6.0. .Their medium con-F tamed a preponderance ofpotassium salts andto 8.6.' But the abundancy oi the hyphae, the

o'nly tracesof calcium and iron salts. It may a,1so,soa

growth associated with better nutrition.

be noted in Table 2 that potassium carbonate is especially toxic tomycelial growth and that even though the pH was 8.1 in bottle #25,'thepresence of 3% potassium carbonate produced only a slight growth. Anycorrelation of pH and rapidity of growth must takeinto consideration thepresence or absence of certain constituents in the substrate.

Yet there may be some basis for deciding what pH is most satisfactory.For example, I have determined that the pH of the mycelium both in thestem and in the cap of the mushroom is 6.6 to 6.7 by both thequinhydrone and the glass electrodes. It is not possible to determinethe pH of the mycelium in the substrate apart from the substrate, but itwould not be far fetched to suppose that the pH of the mycelium in thesubstrate is the same as that of the mycelium in the stemv and cap ofthe mushroom, since there is so little differentiation of tissue in thisfungus.

It may be noted in Table 2 that where the mycelial growth was mostdense, the pH of the spawn was in almost every case 6.6 to 6.7. Otherconditions may bring about a pH of this range in the spawn. But wherethe spawn is most dense and the pH is in this range, such spawn may beconsidered as of the best quality. The pH to be desired in a substratewould then be the one which would result in a pH of 6.6 to 6.7

' and alkali earth elements.

in the spawn, because that pH range exists in the mycelium of themushroom itself. I

The beneficial effects of the salts of such elements as AL'Fe, Cr, Mn,etc. is explained as antagonizing or buffering the effect of the alkaliThe addition of such alkalies as Ca and Mg is to precipitate the aciditycaused by the growth of the mycelium. Although this explanation may notbe entirely correct, the benefits to be derived and the practicality oftheir utilization have been realized.

The heavy incrustation of crystals on the hyphae may be observed when acombination of salts, as disclosed in my method is employed, whereasthere is comparatively no such incrustation and precipitation ofcrystals in the check bottles where no salts were added. It might becontended that this crystalline incrustation is largely responsible forthe milky white, opaque appearance of the spawn prepared by my methed,and not a more abundant mycelial growth and a more complete penetrationof the substrate. However, it has been determined that when thiscombination of salts are added to manure in the ordinary mushroom bed,the carbon dioxide evolution is three times as great as where no saltshave been added. A greater evolution of carbon dioxide can only beinterpreted as 7 caused by a more vigorous growth and a betterpenetration of the substrate. According to some authorities on thebiochemistry of other fungi (R. A. Steinberg, Bull. Torrey Club, 61:241-8, 1934) the translucent and slimy hyphae may be associated withlack of growth due to poor nutrition and to injury by chemicals. and anopaque thermore, the yield of mushrooms is very significantly greaterfrom the beds to which salts have been added.

Another benefit to be derived from the employment of a combination ofthese salts is that no strands develop in the spawn bottle, but'arealways present in bottles to which no such salts in the properproportions have been added, especially in the neck of the bottle.Strands develop only when the substrate becomes increasingly unfavorablefor mycelial growth. On the other hand, the mycelium in the bottlesprepared according to my method is always very fine and very dense.

While these data and experiments were carried out with Agaricuscampestris L., the method described would improve the mycelial growth ofotheredible mushrooms. The mineral compositions of the common ediblemushrooms are very similar, and their reactivity toward the substratemay also be similar. a

By the use of the term substrate, medium, compost, or bed, I intend toinclude any development-supporting material or body suitable for thedevelopment andgrowth of mycelium and also of the mushrooms.

What I claim is: g i

1. The method of treating a mushroom substrate containing a solublealkali substance sufiicient to remove acid conditions arising frommycelial growth, which consists in mixing therewith a salt of a metalselected from the group consisting of Fe, Al, Mn, Cr and Zn in amountsufiicient to buffer and prevent the alkali from injuriously affectingthe mycelial growth and which will react with said alkali to improvesaid growth.

2. The method of treating a mushroom substrate containing 4% to 6% oflime to remove acid conditions arising from mycelial growth, whichconsists in mixing therewith a salt of a metal of the group consistingof Fe, Al, Mn. Cr and Zn in amount sufficient to buffer and prevent thealkali from injuriously affecting the mycelial growth and which willreact with said alkali to improve said growth. I

3. A substrate for mushroom culture containing a soluble alkalisubstance sufficient to rea move acid conditions arising fromv mycelialgrowth, and a salt of a metal selected from the group consisting ofFe,Al, Mn, Cr and Zn in amount sufllcient to buffer and prevent thealkali from injuriously aifecting the said mycelial growth and to reactwith said alkali to improve said growth.

4. A substrate for mushroom culture containing 4% to 6% of lime and asalt of a metal selected from the group consisting of Fe, Al, Mn, Cr andZn in amount sumcient to buffer and prevent the lime from iniuriouslyaflecting the said mycelial growth andto react with said lime to improvesaid growth.

' BENJAMIN B. BTOIIER.

